Fractionated radiotherapy with ionizing radiation (IR) is a mainstay of HNSCC treatment but fails in two-thirds of cases due, in large part, to development of IR resistance. New insights into the molecular basis for IR resistance are needed to develop agents capable of overcoming it. Recently an IR-resistant HNSCC cell line, Nu-61, was developed by serial transplantation and irradiation of xenograft tumors starting with the IR-sensitive HNSCC cell line, SCC-61. Comparison of the gene expression profiles of the initial and final cell lines identified 25 IFN/Stat1 pathway signature genes as candidates for acquired radioresistance. Silencing Stat1 in Nu-61 decreased expression of these genes and rendered these cells IR sensitive, whereas overexpression of Stat1 in the radiosensitive SCC-61 parental cells increased expression of these genes and converted them to IR resistant. We demonstrated that the same set of signature genes is upregulated in xenografts from another IR- resistant cell line, UM-SCC-17B, which further supports the hypothesis that the IFN/Stat1 pathway may be critical for radioresistance and worth targeting in HNSCC. IR resistance in HNSCC also has been linked to Stat3 activation via increased EGFR or LIF-signaling. To develop new strategies for reversing radioresistance, our group used computer-based docking and lead-compound optimization strategies to identify C188-9, a potent, non-toxic, and orally bioavailable dual inhibitor of Stat3 and Stat1. C188-9 potently inhibited growth of tumor xenografts of the IR-resistant HNSCC cell line, UM-SCC-17B. RNA-seq analysis of these xenografts revealed that C188-9 modulated 91 genes regulated directly by Stat3 and/or Stat1 including 23 of the 25 genes in the IFN/Stat1 signature set, many of which also are known to be co-regulated by Stat3. In this proposal, we hypothesize that Stat1 and Stat3 contribute to IR resistance and that use of the dual active Stat1/3 inhibitor, C188-9, as an adjuvant with IR will be more effective at shrinking HNSCC tumor xenografts than IR alone. We have outlined two focused Specific Aims to interrogate these hypotheses. In Aim 1, we will determine the effect in vitro of IR with or without C188-9 on the growth, Stat1 and Stat3 activity, and expression of IFN/Stat1 signature genes in both IR-resistant HNSCC cell lines (UM-SCC-17B, SCC-35, and Nu-61) and IR-sensitive HNSCC cell lines (SCC-61, SCC-15, SCC-9). In Aim 2, we will determine the effect in vivo of IR with or without C188-9 on growth, Stat1 and Stat3 activity, and expression of IFN/Stat1 signature genes in IR-resistant HNSCC xenografts. The combination of IR with a non-toxic agent, such as C188-9, that molecularly targets one or more critical mediator of radioresistance has yet to reach the clinic. Successful completion of this project will allow us to assess whether dual targeting of Stat1 and 3 is a potentially useful strategy for overcoming radioresistance in HNSCC. If successful, these studies will form the preclinical basis for performing clinical trials of C188-9 as an adjuvant to IR in HNSCC to reduce emergence of IR resistance and improve cure rates.